Use of adipose tissue-derived stromal stem cells in treating fistula

ABSTRACT

Provided herein are novel methods and composition utilizing adipose tissue-derived stromal stem cells for treating fistulae.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/993,859, filed on Dec. 24, 2009, which is filed under the provisionsof 35 U.S.C. §371 and claims the priority of International PatentApplication No. PCT/EP2006/004605, filed on May 16, 2006, which iscontinuation of U.S. patent application Ser. No. 11/167,061, filed onJun. 24, 2005, the contents of which are herein specificallyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Generally, a fistula is an abnormal connection or passageway betweenorgans or vessels that normally do not connect. Fistulae can develop invarious parts of the body. For example, types of fistulae, named for theareas of the body in which they occur, include anorectal fistula orfistula-in-ano or fecal fistula (between the rectum or other anorectalarea and the skin surface), arteriovenous fistula or A-V fistula(between an artery and vein), biliary fistula (between the bile ducts tothe skin surface, often caused by gallbladder surgery), cervical fistula(abnormal opening in the cervix), craniosinus fistula (between theintracranial space and a paranasal sinus), enteroenteral fistula(between two parts of the intestine), enterocutaneous fistula (betweenthe intestine and the skin surface, namely from the duodenum or thejejunum or the ileum), enterovaginal fistula (between the intestine andthe vagina), gastric fistula (between the stomach to the skin surface),metroperitoneal fistula (between the uterus and peritoneal cavity),perilymph fistula (a tear between the membranes between the middle andinner ears), pulmonary arteriovenous fistula (between an artery and veinof the lungs, resulting in shunting of blood), rectovaginal fistula(between the rectum and the vagina), umbilical fistula (between theumbilicus and gut), tracheoesophageal fistula (between the breathing andthe feeding tubes) and vesicovaginal fistula (between the bladder andthe vagina). Causes of fistulae include trauma, complications frommedical treatment and disease.

Treatment for fistulae varies depending on the cause and extent of thefistula, but generally involves surgical intervention. Various surgicalprocedures are commonly used, most commonly fistulotomy, placement of aseton (a cord that is passed through the path of the fistula to keep itopen for draining), or an endorectal flap procedure (where healthytissue is pulled over the internal side of the fistula to keep feces orother material from reinfecting the channel). Surgery for anorectalfistulae is not without side effects, including recurrence, reinfection,and incontinence.

Inflammatory bowel diseases, such as Crohn's disease and ulcerativecolitis, are the leading causes of anorectal, enteroenteral, andenterocutaneous fistulae. The reported incidence of fistula in Crohn'sdisease ranges from 17% to 50%. Management of fistulae in patients withCrohn's disease continues to present an extremely challenging problemsince many such fistulae do not respond to available treatments. Suchfistulae and their recurrence are a very distressing complication thatsignificantly reduces the quality of life of affected patients. Recentimprovements in medical treatment (e.g., treatment with Infliximab) andexpert surgical management have decreased the need for complicatedsurgery. However, many patients are not cured. Failure of fistulae toheal is probably due to the suboptimal quality of tissues that have beenaffected by Crohn's disease. Indeed, Crohn's fistulae provide a modelsystem for wound healing under some of the worst possible conditions.

Another leading cause of fistulae is trauma, e.g. by rape, or byinjuries sustained during childbirth, to the tissues of the vagina andthe bladder and/or rectum leading to rectovaginal fistula andvesicovaginal fistula. Every year approximately 100,000 women across thedeveloping world sustain such fistulae (also known as obstetricfistulae) during obstructed labor. During obstructed labor, the pressureof the baby's head against the mother's pelvis cuts off blood supply todelicate tissues in the region. The dead tissue falls away and the womanis left with a vesicovaginal fistula and sometimes a rectovaginalfistula. This hole results in permanent incontinence of urine and/orfeces. The United Nations Population Fund (UNFPA) estimates the world'spopulation of obstetric fistula sufferers at more than two million. Thiscalculation could be a significant underestimate. Success rates forprimary surgical repair range from 88 to 93 percent but decrease withsuccessive attempts. Thus, a significant percentage of women haveobstetrical fistulae that cannot be repaired surgically.

New therapies for fistulae are needed.

SUMMARY OF THE INVENTION

Provided herein, among other things, are novel adipose tissue-derivedstromal stem cell-containing compositions. The adipose tissue-derivedstromal stem cell-containing compositions described herein have adistinct phenotype and exhibit greater homogeneity of phenotype thanpreviously described adipose tissue-derived stromal stem cellcompositions, thus making them more suitable for use in treatingfistulae and wounds than previously described compositions. The adiposetissue-derived stromal stem cell-containing compositions may beformulated with solutions or other substances to serve aspharmaceuticals or medical devices, e.g., as sutures or adhesives.Further, provided are novel methods of treating fistulae and woundsusing adipose tissue-derived stromal stem cells, as well as kits for thepractice of the same.

These embodiments of the present invention, other embodiments, and theirfeatures and characteristics will be apparent from the description,drawings, and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the results of characterization of cells isolated by themethods of Example 1 by immunofluorescence staining. The frequency ofimmunopositive cells is indicated as follows: −, less than 5%; +/−,6-15%; +, 16-50%; ++, 51-85%; and +++, 86-100%. P, Passage number.

FIG. 2 depicts indirect immunofluorescence characterization of adiposetissue-derived stromal stem cells. Celts from patient #001 were passaged6 cells subsequent to implant no. 6. Blue color indicates DAPI-stainednuclei. (A) CD90; (B) c-Kit; and (C) vimentin.

FIG. 3 summarizes the clinical results obtained using certain methodsand compositions of the invention. F, Female; M, male; NI, No implant;NA, Not analyzed.

FIG. 4 depicts growth curves of lipoaspirate-derived cells at differentconcentrations of FBS (0.5, 2.5 and 10%, as indicated). Human synovialfibroblasts were cultured in the presence of either 5% or 10% FBS. Cellnumbers±SD are shown in terms of absorbance at 595 nm. Data are from arepresentative experiment with triplicate wells.

FIG. 5 depicts the blister in the rectal mucosa after cells had beeninjected close to the sutured internal opening.

FIG. 6 depicts photographs of a fistula before (A) and eight weeks after(B) injection of cells.

FIG. 7A depicts histograms of fluorescence immunocytometry correspondingto the profile of surface markers (CD3, CD9, CD10, CD11b, CD13, CD14,CD15, CD16, CD18, CD19, CD28, CD29, CD31, CD34, CD36, CD38, CD44, CD45,CD49a, CD49b, CD49c, CD49d, CD49e and CD49f) obtained from cellsisolated from liposuction samples of a patient involved in the study, atpassage 6.

FIG. 7B depicts histograms of fluorescence immunocytometry correspondingto the profile of surface markers (CD50, CD51, CD54, CD55, CD56, CD58,CD59, CD61, CD62E, CD62L, CD62P, CD90, CD95, CD102, CD104, CD105, CD106,CD 133, CD166, glicoforina, β2 microglobuline, HLA I, HLA II and NGFR)obtained from cells isolated from liposuction samples of a patientinvolved in the study, at passage 6.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention belongs.

The articles “a” and “an” refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

By “adipose tissue” is meant any fat tissue. The adipose tissue may bebrown or white adipose tissue, derived from subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site.Preferably, the adipose tissue is subcutaneous white adipose tissue.Such cells may comprise a primary cell culture or an immortalized cellline. The adipose tissue may be from any organism having fat tissue.Preferably, the adipose tissue is mammalian, most preferably the adiposetissue is human. A convenient source of adipose tissue is fromliposuction surgery, however, the source of adipose tissue or the methodof isolation of adipose tissue is not critical to the invention. Ifstromal cells are desired for autologous transplantation into a subject,the adipose tissue will be isolated from that subject.

“Adipose tissue-derived stromal stem cells” refers to mesenchymal stemcells that originate from adipose tissue.

The term “adhesive” refers to any substance that unites or bondssurfaces together; e.g., a glue.

The term “cellular composition” refers to a preparation of cells, whichpreparation may include, in addition to the cells, non-cellularcomponents such as cell culture media, e.g. proteins, amino acids,nucleic acids, nucleotides, co-enzyme, anti-oxidants, metals and thelike. Furthermore, the cellular composition can have components which donot affect the growth or viability of the cellular component, but whichare used to provide the cells in a particular format, e.g., as polymericmatrix for encapsulation or a pharmaceutical preparation.

The term “culture” refers to any growth of cells, organisms,multicellular entities, or tissue in a medium. The term “culturing”refers to any method of achieving such growth, and may comprise multiplesteps. The term “further culturing” refers to culturing a cell,organism, multicellular entity, or tissue to a certain stage of growth,then using another culturing method to bring said cell, organism,multicellular entity, or tissue to another stage of growth. A “cellculture” refers to a growth of cells in vitro. In such a culture, thecells proliferate, but they do not organize into tissue per se. A“tissue culture” refers to the maintenance or growth of tissue, e.g.,explants of organ primordial or of an adult organ in vitro so as topreserve its architecture and function. A “monolayer culture” refers toa culture in which cells multiply in a suitable medium while mainlyattached to each other and to a substrate. Furthermore, a “suspensionculture” refers to a culture in which cells multiply while suspended ina suitable medium. Likewise, a “continuous flow culture” refers to thecultivation of cells or explants in a continuous flow of fresh medium tomaintain cell growth, e.g. viability. The term “conditioned media”refers to the supernatant, e.g. free of the cultured cells/tissue,resulting after a period of time in contact with the cultured cells suchthat the media has been altered to include certain paracrine and/orautocrine factors produced by the cells and secreted into the culture. A“confluent culture” is a cell culture in which all the cells are incontact and thus the entire surface of the culture vessel is covered,and implies that the cells have also reached their maximum density,though confluence does not necessarily mean that division will cease orthat the population will not increase in size.

The term “culture medium” or “medium” is recognized in the art, andrefers generally to any substance or preparation used for thecultivation of living cells. The term “medium”, as used in reference toa cell culture, includes the components of the environment surroundingthe cells. Media may be solid, liquid, gaseous or a mixture of phasesand materials. Media include liquid growth media as well as liquid mediathat do not sustain cell growth. Media also include gelatinous mediasuch as agar, agarose, gelatin and collagen matrices. Exemplary gaseousmedia include the gaseous phase that cells growing on a petri dish orother solid or semisolid support are exposed to. The term “medium” alsorefers to material that is intended for use in a cell culture, even ifit has not yet been contacted with cells. In other words, a nutrientrich liquid prepared for bacterial culture is a medium. Similarly, apowder mixture that when mixed with water or other liquid becomessuitable for cell culture, may be termed a “powdered medium”. “Definedmedium” refers to media that are made of chemically defined (usuallypurified) components. “Defined media” do not contain poorlycharacterized biological extracts such as yeast extract and beef broth.“Rich medium” includes media that are designed to support growth of mostor all viable forms of a particular species. Rich media often includecomplex biological extracts. A “medium suitable for growth of a highdensity culture” is any medium that allows a cell culture to reach anOD600 of 3 or greater when other conditions (such as temperature andoxygen transfer rate) permit such growth. The term “basal medium” refersto a medium which promotes the growth of many types of microorganismswhich do not require any special nutrient supplements. Most basal mediagenerally comprise of four basic chemical groups: amino acids,carbohydrates, inorganic salts, and vitamins. A basal medium generallyserves as the basis for a more complex medium, to which supplements suchas serum, buffers, growth factors, lipids, and the like are added.Examples of basal media include, but are not limited to, Eagles BasalMedium, Minimum Essential Medium, Dulbecco's Modified Eagle's Medium,Medium 199, Nutrient Mixtures Ham's F-10 and Ham's F-12, McCoy's 5A,Dulbecco's MEM/F-I 2, RPMI 1640, and Iscove's Modified Dulbecco's Medium(IMDM).

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “differentiation” refers to the formation of cells expressingmarkers known to be associated with cells that are more specialized andcloser to becoming terminally differentiated cells incapable of furtherdivision or differentiation. For example, in a pancreatic context,differentiation can be seen in the production of islet-like cellclusters containing an increased proportion of beta-epithelial cellsthat produce increased amounts of insulin. The terms “further” or“greater” differentiation refers to cells that are more specialized andcloser to becoming terminally differentiated cells incapable of furtherdivision or differentiation than the cells from which they werecultured. The term “final differentiation” refers to cells that havebecome terminally differentiated cells incapable of further division ordifferentiation.

The term “fistula” refers to any abnormal passage or communication orconnection, usually between two internal organs or leading from aninternal organ to the surface of the body. Examples of fistulae include,but are not limited to, anorectal fistula or fistula-in-ano or fecalfistula, arteriovenous fistula or A-V fistula, biliary fistula, cervicalfistula, craniosinus fistula, enteroenteral fistula, enterocutaneousfistula, enterovaginal fistula, gastric fistula, metroperitoneal fistulaperilymph, pulmonary arteriovenous fistula, rectovaginal fistula,umbilical fistula, tracheoesophageal fistula and vesicovaginal fistula.

The term “including” is used herein to mean “including but not limitedto”. “Including” and “including but not limited to” are usedinterchangeably.

“Marker” refers to a biological molecule whose presence, concentration,activity, or phosphorylation state may be detected and used to identifythe phenotype of a cell.

A “patch” is a dressing or covering applied to cover or protect a woundor other sore.

A “patient”, “subject” or “host” to be treated by the subject method maymean either a human or non-human animal.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. The term “phenotype” refers to the observable characteristicsof a cell, such as size, morphology, protein expression, etc.

The term “progenitor cell” refers to a cell that has the capacity tocreate progeny that are more differentiated than itself. For example,the term may refer to an undifferentiated cell or cell differentiated toan extent short of final differentiation which is capable ofproliferation and giving rise to more progenitor cells having theability to generate a large number of mother cells that can in turn giverise to differentiated, or differentiable daughter cells. In a preferredembodiment, the term progenitor cell refers to a generalized mother cellwhose descendants (progeny) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. By thisdefinition, stem cells may also be progenitor cells, as well as the moreimmediate precursors to terminally differentiated cells.

“Proliferation” refers to an increase in cell number. “Proliferating”and “proliferation” refer to cells undergoing mitosis.

As used herein, the term “solution” includes a pharmaceuticallyacceptable carrier or diluent in which the cells of the invention remainviable.

The term “substantially pure”, with respect to adipose tissue-derivedstem cell populations, refers to a population of adipose tissue-derivedstem cell cells that is at least about 75%, preferably at least about85%, more preferably at least about 90%, and most preferably at leastabout 95% pure, with respect to adipose tissue-derived stromal stemcells making up a total cell population. Recast, the term “substantiallypure” refers to a population of adipose tissue-derived stromal stemcells of the present invention that contain fewer than about 20%, morepreferably fewer than about 10%, most preferably fewer than about 5%, oflineage committed cells in the original unamplified and isolatedpopulation prior to subsequent culturing and amplification.

“Support” as used herein refers to any device or material that may serveas a foundation or matrix for the growth of adipose tissue-derivedstromal stem cells.

The term “suture” refers to a thread or fiber or other fasteningmaterial that can be used to sew a wound together.

The term “treating” as used herein refers to repairing a fistula orwound, as well as preventing a fistula or wound from worsening orrecurring.

“Therapeutic agent” or “therapeutic” refers to an agent capable ofhaving a desired biological effect on a host. Chemotherapeutic andgenotoxic agents are examples of therapeutic agents that are generallyknown to be chemical in origin, as opposed to biological, or cause atherapeutic effect by a particular mechanism of action, respectively.Examples of therapeutic agents of biological origin include growthfactors, hormones, and cytokines. A variety of therapeutic agents areknown in the art and may be identified by their effects. Certaintherapeutic agents are capable of regulating cell proliferation anddifferentiation. Examples include chemotherapeutic nucleotides, drugs,hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g.,antisense oligonucleotides that bind to a target nucleic acid sequence(e.g., mRNA sequence)), peptides, and peptidomimetics.

A “wound” is an injury or damage to tissue, caused by physical means,causing disruption of normal continuity of the tissue.

2. Novel Adipose Tissue-Derived Stromal Stem Cell-ContainingCompositions

In one aspect, the invention relates to adipose tissue-derived stromalstem cell-containing compositions with certain characteristics, such asa particular phenotype. For example, the adipose tissue-derived stromalstem cells in a cellular composition of the invention may becharacterized by cell surface marker expression, size, glucoseconsumption, lactate production, and cell yield/viability. Yet anotheraspect of the present invention concerns adipose tissue-derived stromalstem cell-containing compositions which include, as a cellularcomponent, substantially pure preparations of adipose tissue-derivedstromal stem cells having a particular phenotype, or the progenythereof. Adipose tissue-derived stromal stem cell-containingcompositions of the present invention include not only substantiallypure populations of the progenitor cells, but can also include cellculture components, e.g., culture media including amino acids, metals,coenzyme factors, as well as small populations of other stromal cells,e.g., some of which may arise by subsequent differentiation of cells ofthe invention. Furthermore, other non-cellular components can includethose which render the cellular component suitable for support underparticular circumstances, e.g., implantation, e.g., continuous culture,or suitable for use as a biomaterial or pharmaceutical composition.

In certain embodiments, the adipose tissue-derived stromal stemcell-containing compositions are produced through the culture methodsdescribed in Section 4 and the Exemplification.

In one embodiment, provided is an adipose tissue-derived stromal stemcell-containing composition, wherein at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% or preferably at least about 96%,97%, 98% or 99% of the stem cells express the CD9, CD 10, CD13. CD29,CD44, CD49A. CD51, CD54, CD55, CD58, CD59, CD90 and/or CD105 markers. Incertain embodiments of the adipose tissue-derived stromal stemcell-containing compositions, fewer than about 15%, about 10%, about 5%,and preferably about 4%, 3%, 2% or 1% of the stem cells express the CD34CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106and/or CD133 markers.

In another embodiment, provided is an adipose tissue-derived stromalstem cell-containing composition, wherein at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95% or preferably at least about 96%,97%, 98% or 99% of the stem cells express the c-Kit, vimentin and/orCD90 markers. In certain embodiments of the adipose tissue-derivedstromal stem cell-containing compositions, fewer than about 15%, about10%, about 5%, and preferably about 4%, 3%, 2% or 1% of the stem cellsexpress the CD34, Factor VIII, alpha-actin, desmin, S-100 and/or keratinmarkers. Also provided is an adipose tissue-derived stromal stem cellpopulation that express the c-Kit, vimentin and CD90 markers and doesnot express the CD34, Factor VIII, alpha-actin, desmin, S-100 andkeratin markers.

The phenotypic characterization of a cell population by surface markerscan be performed either by individual staining of the cells (flowcytometry) or by making histological cuts of the population in situ,done in accordance with normal methods. The determination of the profileof expression of surface markers by antibodies, immunophenotypecharacterization, may be direct, using a labeled antibody or indirect,using a second labeled antibody against the primary specific antibody ofthe cell marker, thus achieving signal amplification. On the other hand,the presence or absence of binding to the antibody may be determined bydifferent methods that include but are not limited to immunofluorescencemicroscopy and radiography. Similarly, it is possible to carry out themonitoring of the levels of binding of the antibody by flow cytometry, atechnique that allows the levels of fluorochrome to be correlated withthe quantity of antigens present on the cell surface bound specificallyto the labeled antibodies. The differential expression of a series ofsurface markers on a cell population provides a method foridentification and isolation of said population.

In certain embodiments, the adipose tissue-derived stromal stemcell-containing compositions are suspensions of adipose tissue-derivedstromal stem cells in various solutions or materials, e.g. for use aspharmaceuticals or biomaterials, as described in more detail below. Inone embodiment, the cellular composition comprises a suspension of thesubject adipose tissue-derived stromal stem cells in Ringer's solutionand HSA. In another embodiment, the cellular composition comprises asuspension of the subject adipose tissue-derived stromal stem cells in amaterial, such as a polymer, glue, gel, etc. Such suspensions may beprepared, for example, by sedimenting out the subject adiposetissue-derived stromal stem cells from the culture medium andre-suspending them in the desired solution or material. The cells may besedimented and/or changed out of the culture medium, for example, bycentrifugation, filtration, ultrafiltration, etc.

The concentration of the subject adipose tissue-derived stromal stemcells in the subject adipose tissue-derived stromal stem cell-containingcompositions may be at least about 5×10⁶ cells/mL, at least about 10×10⁶cells/mL, at least about 20×10⁶ cells/mL, at least about 30×10⁶cells/mL, or at least about 40×10⁶ cells/mL.

Accordingly, another aspect of the present invention pertains to theprogeny of the subject adipose tissue-derived stromal stem cells, e.g.those cells which have been derived from the adipose tissue-derivedstromal stem cells. Such progeny can include subsequent generations ofadipose tissue-derived stromal stem cells, as well as lineage committedcells generated by inducing differentiation of the subject adiposetissue-derived stromal stem cells after their isolation from theexplant, e.g., induced in vitro. In certain embodiments, the progenycells are obtained after about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, or about 10 passages from the parentalpopulation. However, the progeny cells may be obtained after any numberof passages from the parental population.

In certain embodiments, the adipose tissue-derived stromal stemcell-containing compositions of the invention will be provided as partof a pharmaceutical preparation, e.g., a sterile, free of the presenceof unwanted virus, bacteria and other pathogens, as well as pyrogen-freepreparation. That is, for human administration, the subject compositionsshould meet sterility, pyrogenicity as well as general safety and puritystandards as required by FDA Office of Biologics standards.

In certain embodiments, such adipose tissue-derived stromal stemcell-containing compositions can be used for transplantation intoanimals, preferably mammals, and even more preferably humans. The cellscan be preferably autologous, but also allogeneic or xenogeneic withrespect to the transplantation host. Because of difficulties inobtaining sufficient autologous stem cells, adipose tissue-derivedstromal stem cell from allogenic donor could constitute a valuablealternative source of stem cells for therapeutic use. It is known in theart that bone marrow stromal stem cells and adipose tissue-derivedstromal cells did not provoke a response of allogenic lymphocytes invitro and consequently, allogenic adipose tissue-derived stromal stemcells derived from a donor could be theorically used for any patient,irrespective of MHC incompatibility.

Methods of administering the adipose tissue-derived stromal stemcell-containing compositions to subjects, particularly human subjects,which are described in detail herein, include injection or implantationof the cells into target sites in the subjects, the cells can beinserted into a delivery device which facilitates introduction by,injection or implantation, of the cells into the subjects. Such deliverydevices include tubes, e.g., catheters, for injecting cells and fluidsinto the body of a recipient subject. In a preferred embodiment, thetubes additionally have a needle, e.g., a syringe, through which theadipose tissue-derived stromal stem cell-containing compositions can beintroduced into the subject at a desired location. The adiposetissue-derived stromal stem cell-containing compositions can be insertedinto such a delivery device, e.g., a syringe, in different forms. Forexample, the adipose tissue-derived stromal stem cell-containingcompositions include compositions of adipose tissue-derived stromal stemcells that are suspended in a solution or embedded in a support matrixwhen contained in such a delivery device.

Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. The solution ispreferably sterile and fluid to the extent that easy syringabilityexists. Preferably, the solution is stable under the conditions ofmanufacture and storage and preserved against the contaminating actionof microorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. Solutions that are adipose tissue-derived stromal stem cellcompositions of the invention can be prepared by incorporating adiposetissue-derived stromal stem cells as described herein in apharmaceutically acceptable carrier or diluent and, as required, otheringredients enumerated above, followed by filtered sterilization.

Some examples of materials and solutions which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

In certain embodiments, the adipose tissue-derived stromal stemcell-containing compositions further comprise an adhesive. In certainembodiments, the adhesive is a fibrin-based adhesive, such as a fibringel or fibrin glue or fibrin-based polymer or adhesive, or other tissueadhesive or surgical glue, such as, for example cyanoacrylate, collagen,thrombin, and polyethylene glycol. Other materials that may be usedinclude but are not limited to calcium alginate, agarose, types I, II,IV or other collagen isoform, poly-lactic/poly-glycolic acid,hyaluronate derivatives or other materials (Perka C. et al. (2000) J.Biomed. Mater. Res. 49:305-311; Sechriest V F. et al. (2000) J. Biomed.Mater. Res. 49:534-541; Chu C R et al. (1995) J. Biomed. Mater. Res.29:1147-1154; Hendrickson D A et al. (1994) Orthop. Res. 12:485-497). Inother embodiments, the adhesive is a liquid bandage, wherein adiposetissue-derived stromal stem cell-containing compositions of the methodare mixed with the liquid bandage material. A “liquid bandage” is asolution comprising a compound, e.g. a polymeric material, which isapplied to a wound with a spray or a brush, followed by removing thesolvent by vaporization to provide a protective film on the wound.

Provided herein are also methods for preparing adipose tissue-derivedstromal stem cell-containing compositions comprising compounds ormaterials for use in repairing fistula or wounds. In one embodiment, amethod of preparing such materials comprises suspending the adiposetissue-derived stromal stem cells of a subject cellular composition withthe material. In one embodiment, the adipose tissue-derived stromal stemcells are sedimented out of the culture medium and re-suspended in afibrin glue or gel. Fibrin glues and gels and other fibrin-basedpolymers and adhesives are well-known in the art and are commerciallyavailable. For example, a commercially available fibrin glue kit is theTISSUCOL® Duo 2.0, and other commercially available fibrin sealantsinclude CROSSEAL®, TISSEEL VH® Fibrin Sealant, and the like.

The adipose tissue-derived stromal stem cell-containing compositions ofthe invention may also be used to coat a support, e.g. a medical device.For example, the support may be a suture, thread, meniscus repairdevice, rivet, tack, staple, screw, bone plate, bone plating system,surgical mesh, patch, e.g. a repair patch, cardiovascular patch, orpericardial patch, sling, orthopedic pin, adhesion barrier, stent,guided tissue repair/regeneration device, articular cartilage repairdevice, nerve guide, tendon repair device, atrial septal defect repairdevice, bulking or filling agent, vein valve, bone marrow scaffold,meniscus regeneration device, ligament and tendon graft, ocular cellimplant, spinal fusion cage, skin substitute, dural substitute, bonegraft substitute, bone dowel, wound dressing, glue, polymer or hemostat.

Supports into which the adipose tissue-derived stromal stemcell-containing compositions can be incorporated or embedded or ontowhich the adipose tissue-derived stromal stem cell-containingcompositions may be coated include matrices which arerecipient-compatible and which degrade into products which are notharmful to the recipient. Natural and/or synthetic biodegradablematrices are examples of such matrices. Natural biodegradable matricesinclude plasma clots, e.g., derived from a mammal, and collagenmatrices. Synthetic biodegradable matrices include synthetic polymerssuch as polyanhydrides, polyorthoesters, and polylactic acid. Otherexamples of synthetic polymers and methods of incorporating or embeddingcells into these matrices are known in the art. See e.g., U.S. Pat. No.4,298,002 and U.S. Pat. No. 5,308,701. These matrices provide supportand protection for the fragile cells in vivo.

The support may be coated with cells in any way as known to one of skillin the art, e.g. by soaking, spraying, painting, imprinting, etc.

In one embodiment, the support is a suture, staple, absorbable thread,non-absorbable thread, natural thread, synthetic thread, monofilamentthread or multifilament thread (also called braids). Preferred methodsof preparing sutures and other supports used to close wounds coated withadipose tissue-derived stromal stem cells are disclosed in U.S. patentapplication Ser. No. 11/056,241 “Biomaterial for Suturing”, filed Feb.14, 2005, which application is incorporated by reference in itsentirety. The adipose tissue-derived stromal stem cell-containingcompositions disclosed herein represent novel compositions that may beused with the methods disclosed in U.S. patent application Ser. No.11/056,241.

Further, in any of the adipose-tissue derived stromal stemcell-containing compositions, at least one therapeutic agent may beincorporated into the composition. For example, a composition maycontain an analgesic, to aid in treating inflammation or pain at thesite of the fistula or wound, or an anti-infective agent to preventinfection of the site treated with the composition.

More specifically, non-limiting examples of useful therapeutic agentsinclude the following therapeutic categories: analgesics, such asnonsteroidal anti-inflammatory drugs, opiate agonists and salicylates;anti-infective agents, such as antihelmintics, antianaerobics,antibiotics, aminoglycoside antibiotics, antifungal antibiotics,cephalosporin antibiotics, macrolide antibiotics, miscellaneous β-lactamantibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamideantibiotics, tetracycline antibiotics, antimycobacterials,antituberculosis antimycobacterials, antiprotozoals, antimalarialantiprotozoals, antiviral agents, anti-retroviral agents, scabicides,anti-inflammatory agents, corticosteroid anti-inflammatory agents,antipruritics/local anesthetics, topical anti-infectives, antifungaltopical anti-infectives, antiviral topical anti-infectives; electrolyticand renal agents, such as acidifying agents, alkalinizing agents,diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics,osmotic diuretics, potassium-sparing diuretics, thiazide diuretics,electrolyte replacements, and uricosuric agents; enzymes, such aspancreatic enzymes and thrombolytic enzymes; gastrointestinal agents,such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatoryagents, salicylate gastrointestinal anti-inflammatory agents, antacidanti-ulcer agents, gastric acid-pump inhibitor anti-ulcer agents,gastric mucosal anti-ulcer agents, H2-blocker anti-ulcer agents,cholelitholytic agents, digestants, emetics, laxatives and stoolsofteners, and prokinetic agents; general anesthetics, such asinhalation anesthetics, halogenated inhalation anesthetics, intravenousanesthetics, barbiturate intravenous anesthetics, benzodiazepineintravenous anesthetics, and opiate agonist intravenous anesthetics;hormones and hormone modifiers, such as abortifacients, adrenal agents,corticosteroid adrenal agents, androgens, anti-androgens, immunobiologicagents, such as immunoglobulins, immunosuppressives, toxoids, andvaccines; local anesthetics, such as amide local anesthetics and esterlocal anesthetics; musculoskeletal agents, such as anti-goutanti-inflammatory agents, corticosteroid anti-inflammatory agents, goldcompound anti-inflammatory agents, immunosuppressive anti-inflammatoryagents, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylateanti-inflammatory agents, minerals; and vitamins, such as vitamin A,vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

Preferred classes of useful therapeutic agents from the above categoriesinclude: (1) analgesics in general, such as lidocaine or derivativesthereof, and nonsteroidal anti-inflammatory drugs (NSAIDs) analgesics,including diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opiateagonist analgesics, such as codeine, fentanyl, hydromorphone, andmorphine; (3) salicylate analgesics, such as aspirin (ASA) (entericcoated ASA); (4) H₁-blocker antihistamines, such as clemastine andterfenadine; (5) anti-infective agents, such as mupirocin; (6)antianaerobic anti-infectives, such as chloramphenicol and clindamycin;(7) antifungal antibiotic anti-infectives, such as amphotericin b,clotrimazole, fluconazole, and ketoconazole; (8) macrolide antibioticanti-infectives, such as azithromycin and erythromycin; (9)miscellaneous β-lactam antibiotic anti-infectives, such as aztreonam andimipenem; (10) penicillin antibiotic anti-infectives, such as nafcillin,oxacillin, penicillin G, and penicillin V; (11) quinolone antibioticanti-infectives, such as ciprofloxacin and norfloxacin; (12)tetracycline antibiotic anti-infectives, such as doxycycline,minocycline, and tetracycline; (13) antituberculosis antimycobacterialanti-infectives such as isoniazid (INH), and rifampin; (14)antiprotozoal anti-infectives, such as atovaquone and dapsone; (15)antimalarial antiprotozoal anti-infectives, such as chloroquine andpyrimethamine; (16) anti-retroviral anti-infectives, such as ritonavirand zidovudine; (17) antiviral anti-infective agents, such as acyclovir,ganciclovir, interferon alfa, and rimantadine; (18) antifungal topicalanti-infectives, such as amphotericin B, clotrimazole, miconazole, andnystatin; (19) antiviral topical anti-infectives, such as acyclovir;(20) electrolytic and renal agents, such as lactulose; (21) loopdiuretics, such as furosemide; (22) potassium-sparing diuretics, such astriamterene; (23) thiazide diuretics, such as hydrochlorothiazide(HCTZ); (24) uricosuric agents, such as probenecid; (25) enzymes such asRNase and DNase; (26) antiemetics, such as prochlorperazine; (27)salicylate gastrointestinal anti-inflammatory agents, such assulfasalazine; (28) gastric acid-pump inhibitor anti-ulcer agents, suchas omeprazole; (29) H₂-blocker anti-ulcer agents, such as cimetidine,famotidine, nizatidine, and ranitidine; (30) digestants, such aspancrelipase; (31) prokinetic agents, such as erythromycin; (32) esterlocal anesthetics, such as benzocaine and procaine; (33) musculoskeletalcorticosteroid anti-inflammatory agents, such as beclomethasone,betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone;(34) musculoskeletal anti-inflammatory immunosuppressives, such asazathioprine, cyclophosphamide, and methotrexate; (35) musculoskeletalnonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac,ibuprofen, ketoprofen, ketorlac, and naproxen; (36) minerals, such asiron, calcium, and magnesium; (37) vitamin B compounds, such ascyanocobalamin (vitamin B₁₂) and niacin (vitamin B₃); (38) vitamin Ccompounds, such as ascorbic acid; and (39) vitamin D compounds, such ascalcitriol.

In certain embodiments, the therapeutic agent may be a growth factor orother molecule that affects cell differentiation and/or proliferation.Growth factors that induce final differentiation states are well-knownin the art, and may be selected from any such factor that has been shownto induce a final differentiation state. Growth factors for use inmethods described herein may, in certain embodiments, be variants orfragments of a naturally-occurring growth factor. For example, a variantmay be generated by making conservative amino acid changes and testingthe resulting variant in one of the functional assays described above oranother functional assay known in the art. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

As those skilled in the art will appreciate, variants or fragments ofpolypeptide growth factors can be generated using conventionaltechniques, such as mutagenesis, including creating discrete pointmutation(s), or by truncation. For instance, mutation can give rise tovariants which retain substantially the same, or merely a subset, of thebiological activity of a polypeptide growth factor from which it wasderived.

3. Methods of Preparing Novel Adipose Tissue-Derived Stromal StemCell-Containing Compositions

Methods of preparing the adipose tissue-derived stromal stem cellscomprising the above-described adipose tissue-derived stromal stemcell-containing compositions are also provided. In one embodiment, amethod comprises: (a) collecting adipose tissue from a subject; (b)obtaining a cell suspension by enzymatic digestion; (c) sedimenting thecell suspension and resuspending the cells in a culture medium; (d)culturing of the cells for at least about 10 days; and (g) expanding thecells for at least two culture passages.

Preferably, the adipose tissue-derived stromal stem cells are isolatedfrom the adipose tissue of the subject into which the final adiposetissue-derived stromal stem cell-containing compositions are to beintroduced. However, the stromal stem cells may also be isolated fromany organism of the same or different species as the subject. Anyorganism with adipose tissue can be a potential candidate. Preferably,the organism is mammalian, most preferably the organism is human.

In certain embodiments, the cells are cultured for at least about 15, atleast about 20 days, at least about 25 days, or at least about 30 days.It is preferable that cells are expanded in culture longer to improvethe homogeneity of the cell phenotype in the cell population.

In certain embodiments, the cells are expanded in culture for at leastthree culture passages or “passaged at least three times.” In otherembodiments, the cells are passaged at least four times, at least fivetimes, at least six times, at least seven times, at least eight times,at least nine times, or at least ten times. It is preferable that cellsare passaged more than three times to improve the homogeneity of thecell phenotype in the cell population. Indeed, the cells may be expandedin culture indefinitely so long as the homogeneity of the cell phenotypeis improved and differential capacity is maintained.

Cells may be cultured by any technique known in the art for theculturing of stem cells. A discussion of various culture techniques, aswell as their scale-up, may be found in Freshney, R. I., Culture ofAnimal Cells: A Manual of Basic Technique, 4th Edition, Wiley-Liss 2000.In certain embodiments, the cells are cultured by monolayer culture. Inone embodiment, the cells are cultured and passaged as described inExample 1 below.

Any medium capable of supporting stromal cells in tissue culture may beused. Media formulations that will support the growth of fibroblastsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimal Essential Medium (.alpha.MEM), andRoswell Park Memorial Institute Media 1640 (RPMI Media 1640) and thelike. Typically, 0 to 20% Fetal Bovine Serum (FBS) or 1-20% horse serumwill be added to the above media in order to support the growth ofstromal cells and/or chondrocytes. However, a defined medium could beused if the necessary growth factors, cytokines, and hormones in FBS forstromal cells and chondrocytes are identified and provided atappropriate concentrations in the growth medium. Media useful in themethods of the invention may contain one or more compounds of interest,including, but not limited to antibiotics mitogenic or differentiativecompounds for stromal cells. The cells will be grown at temperaturesbetween 31.degree. C. to 37.degree. C. in a humidified incubator. Thecarbon dioxide content will be maintained between 2% to 10% and theoxygen content between 1% and 22%. Cells may remain in this environmentfor periods of up to 4 weeks.

Antibiotics which can supplemented into the medium include, but are notlimited to penicillin and streptomycin. The concentration of penicillinin the chemically defined culture medium is about 10 to about 200 unitsper ml. The concentration of streptomycin in the chemically definedculture medium is about 10 to about 200 ug/ml.

The adipose tissue derived stromal stem cells may be stably ortransiently transfected or transduced with a nucleic acid of interestusing a plasmid, viral or alternative vector strategy. Nucleic acids ofinterest include, but are not limited to, those encoding gene productswhich enhance the production of extracellular matrix components found inthe tissue type to be repaired, e.g. intestinal wall or vaginal wall.

The transduction of viral vectors carrying regulatory genes into thestromal stem cells can be performed with viral vectors (adenovirus,retrovirus, adeno-associated virus, or other vector) purified by cesiumchloride banding or other method at a multiplicity of infection (viralunits:cell) of between 10:1 to 2000:1. Cells will be exposed to thevirus in serum free or serum-containing medium in the absence orpresence of a cationic detergent such as polyethyleneimine orLIPOFECTAMINE™ for a period of 1 hour to 24 hours (Byk T. et al. (1998)Human Gene Therapy 9:2493-2502; Sommer B. et al. (1999) Calcif. TissueInt. 64:45-49).

Other suitable methods for transferring vectors or plasmids into stemcells include lipid/DNA complexes, such as those described in U.S. Pat.Nos. 5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202;and 6,051,429. Suitable reagents include LIPOFECTAMINE™, a 3:1 (w/w)liposome formulation of the poly-cationic lipid2,3-dioleyloxy-N-[2(sperminecarbox-amido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA) (Chemical Abstracts Registry name:N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl}amino)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminiumtrifluoroacetate), and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) in membrane filtered water. Exemplary isthe formulation LIPOFECTAMINE 2000™ (available from Gibco/LifeTechnologies #11668019). Other reagents include: FUGENE™ 6 TransfectionReagent (a blend of lipids in non-liposomal form and other compounds in80% ethanol, obtainable from Roche Diagnostics Corp. #1814443); andLIPOTAXI™ transfection reagent (a lipid formulation from InvitrogenCorp., #204110). Transfection of stem cells can be performed byelectroporation, e.g., as described in M. L. Roach and J. D. McNeish(2002) Methods in Mol. Biol. 185:1. Suitable viral vector systems forproducing stem cells with stable genetic alterations may be based onadenoviruses and retroviruses, and may be prepared using commerciallyavailable virus components.

The transfection of plasmid vectors carrying regulatory genes into thestem stromal cells can be introduced into the cells in monolayercultures by use of calcium phosphate DNA precipitation or cationicdetergent methods (LIPOFECTAMINE™, DOTAP) or in three dimensionalcultures by incorporation of the plasmid DNA vectors directly into thebiocompatible polymer (Bonadio J. et al. (1999) Nat. Med. 5:753-759).

For the tracking and detection of functional proteins encoded by thesegenes, the viral or plasmid DNA vectors will contain a readilydetectable marker gene, such as the green fluorescent protein orbeta-galactosidase enzyme, both of which can be tracked by histochemicalmeans.

4. Methods of Treating Fistulae and Wounds

Another aspect of the invention concerns a novel method for usingadipose tissue-derived stromal stem cells in treating fistulae andwounds. In preferred embodiments, the adipose tissue-derived stromalstem cells are derived from the adipose tissue of the subject to betreated. In other preferred embodiments, the adipose tissue-derivedstromal stem cells comprise a adipose tissue-derived stromal stemcell-containing composition described herein. However, otherpreparations of adipose tissue-derived stromal stem cells may be used inthe methods described herein, e.g. such as those described in U.S. Pat.Nos. 6,777,231 and 6,555,374 and U.S. patent application Ser. No.11/065,461 “Identification and Isolation of Multipotent Cells FromNon-Osteochondral Mesenchymal Tissue”, filed on Feb. 25, 2005.

In one embodiment, a method of treating a fistula in a subjectcomprises: (a) closing the internal hole with a suture and (b)delivering at least about 10×10⁶, at least about 20×10⁶, at least about30×10⁶, or at least about 40×10⁶ adipose tissue-derived stromal stemcells, e.g., in an adipose tissue-derived stromal stem cell-containingcomposition of the invention, to the closed sutured internal hole. Incertain embodiments, e.g., wherein the first delivery of cells isinsufficient, the method may further comprise: (c) delivering a seconddose of at least about 20×10⁶ cells, at least about 30×10⁶, or at leastabout 40×10⁶ adipose tissue-derived stromal stem cells, e.g., in anadipose tissue-derived stromal stem cell-containing composition of theinvention, to the closed sutured internal hole.

In another embodiment, the adipose tissue-derived stromal stemcell-containing composition used in the method is one wherein at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 85%, at least about 90%, at least about 95% or preferablyat least about 96%, 97%, 98% or 99% of the stem cells express the CD9,CD10, CD13. CD29, CD44, CD49A. CD51, CD54, CD55, CD58, CD59 CD90 and/orCD 105 markers.

In another embodiment, the adipose tissue-derived stromal stemcell-containing composition used in the method is one wherein at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 85%, at least about 90%, at least about 95% or preferablyat least about 96%, 97%, 98% or 99% of the stem cells express the c-Kit,vimentin and/or CD90 markers.

Common methods of administering the cells of the present invention tosubjects, particularly human subjects, some of which are described indetail herein, include injection or implantation of the cells intotarget sites in the subjects, the cells of the invention can be insertedinto a delivery device which facilitates introduction by, injection orimplantation, of the cells into the subjects. Such delivery devicesinclude tubes, e.g., catheters, for injecting cells and fluids into thebody of a recipient subject. In a preferred embodiment, the tubesadditionally have a needle, e.g., a syringe, through which the cells ofthe invention can be introduced into the subject at a desired location.The cells of the invention can be inserted into such a delivery device,e.g., a syringe, in different forms. For example, the cells can besuspended in a solution or embedded in a support matrix when containedin such a delivery device. Pharmaceutically acceptable carriers anddiluents include saline, aqueous buffer solutions, solvents and/ordispersion media. The use of such carriers and diluents is well known inthe art. The solution is preferably sterile and fluid to the extent thateasy syringability exists. Preferably, the solution is stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating progenitor cells as described herein in apharmaceutically acceptable carrier or diluent and, as required, otheringredients enumerated above, followed by filtered sterilization.

In other embodiments, a method of treating a fistula in a subjectcomprises: (a) closing the internal hole with a suture that comprisesadipose tissue-derived stromal stem cells, e.g., from a subject adiposetissue-derived stromal stem cell-containing composition. Such suturescoated with cells in the subject adipose tissue-derived stromal stemcell-containing compositions are described in detail. in U.S. patentapplication Ser. No. 11/056,241, filed on Feb. 14, 2005, which isincorporated herein by reference.

The methods may in some embodiments further comprise: (d) deep scrapingof at least one fistula track and (e) filling said fistula track with amaterial. In certain embodiments, the method may further comprisedelivering at least about 10×10⁶ adipose tissue-derived stromal stemcells, e.g., from a subject cellular composition, to the material.Preferably, the material is a fibrin-based polymer or adhesive, such asa fibrin glue or gel. In certain embodiments, the dose of at least about10×10⁶ adipose tissue-derived stromal stem cells is already encompassedwithin the material, e.g., such that the material comprises the adiposetissue-derived stem cell containing-composition.

In a further embodiment, a method of treating a fistula in a subjectcomprises:

-   -   (i) deep scraping of at least one fistula track    -   (ii) closing the internal hole of the scraped track with a        suture    -   (iii) delivering at least about 10×10⁶, at least about 20×10⁶,        at least about 30×10⁶, or at least about 40×10⁶ adipose        tissue-derived stromal stem cells to the closed sutured internal        hole,        e.g., in an adipose tissue-derived stromal stem cell-containing        composition of the invention.

In certain embodiments, e.g., wherein the first delivery of cells isinsufficient, the method may further comprise:

-   -   (iv) delivering a second dose of at least about 20×10⁶ cells, at        least about 30×10⁶, or at least about 40×10⁶ adipose        tissue-derived stromal stem cells to the closed sutured internal        hole,        e.g., in an adipose tissue-derived stromal stem cell-containing        composition of the invention.

Step (i) is preferably carried out by deep scraping all fistula tracksto be treated for example, a curettage needle is introduced in thefistula tract, and an induced bleeding is produced by scraping thefistula walls in order to obtain natural fibrin which will fill thefistula tract. Recent clinical studies by the inventors suggest that thenatural fibrin produced by this scraping method is a preferred optioncompared with the use of artificial fibrin sealants, therefore in apreferred embodiment of the method of the invention the fistular tractsto be treated are not filled with such material.

Step (iv) is preferably carried out by local delivery of the cells, forexample an adipose tissue-derived stromal stem cell-containingcomposition, by injection into the fistula walls along the fistulatract. For example, two injections of 10 million cells along 3 cm offistula tract.

The methods of the invention may be used to treat any fistula, includingbut not limited to anorectal fistula or fistula-in-ano or fecal fistula,arteriovenous fistula or A-V fistula, biliary fistula, cervical fistula,craniosinus fistula, enteroenteral fistula, enterocutaneous fistula,enterovaginal fistula, gastric fistula, metroperitoneal fistulaperilymph, pulmonary arteriovenous fistula, rectovaginal fistula,umbilical fistula, tracheoesophageal fistula and vesicovaginal fistula.Preferably, the methods may be used to treat intestinal fistulae, e.g.those connecting the intestine to itself or to another organ, such asrectovaginal fistula, enteroenteral fistula, enterocutaneous fistula andenterovaginal fistula. In another preferred embodiment, the methods maybe used to treat vaginal or uterine fistulae, e.g. those connecting thevagina or uterus to itself or to another organ, such as cervicalfistula, rectovaginal fistula, enterovaginal fistula, and vesicovaginalfistula.

The fistula may be accessed for surgical repair via any method known inthe art, e.g., via incision, catheter, etc.

In another embodiment, a method of treating a wound in a subjectcomprises: (a) closing the wound with a suture and (b) delivering atleast about 10×10⁶, at least about 20×10⁶, at least about 30×10⁶, or atleast about 40×10⁶ adipose tissue-derived stromal stem cells, e.g., inan adipose-tissue derived stromal stem cell-containing composition, tothe closed sutured wound. In certain embodiments, e.g., wherein thefirst delivery of cells is insufficient, the method may furthercomprise: (c) delivering a second dose of at least about 20×10⁶ cells,at least about 30×10⁶, or at least about 40×10⁶ adipose tissue-derivedstromal stem cells, e.g., in an adipose-tissue derived stromal stemcell-containing composition, to the closed sutured wound. In otherembodiments, the wound may be filled with an adipose-tissue derivedstromal stem cell-containing composition of the invention, e.g. a doseof at least about 10×10⁶ adipose tissue-derived stromal stem cellsencompassed within a material, e.g., such that the material comprisesthe cellular composition, wherein the material is, for example, anadhesive or glue. In other embodiments, a method of treating a wound ina subject comprises: (a) closing the wound with a suture that comprisesadipose tissue-derived stromal stem cells, e.g., from a subjectadipose-tissue derived stromal stem cell-containing composition. Suchsutures coated with cells from the subject adipose tissue-derivedstromal stern cell-containing compositions are described in detail aboveand in U.S. patent application Ser. No. 11/056,241, filed on Feb. 14,2005, which is incorporated herein by reference.

The methods described above may further comprise administering atherapeutic agent to the subject being treated, e.g. systemically orlocally at the site of suturing. In certain embodiments, the adiposetissue-derived stromal stem cells are formulated in an adiposetissue-derived stromal stem cell-containing composition which contains atherapeutic agent, as described above. In other embodiments, thetherapeutic agent is administered separately, e.g. simultaneously withthe methods, before the method is performed, or after the method isperformed. In some embodiments, the therapeutic agent is administered tothe subject before, during and after the methods are performed on thesubject. Exemplary therapeutic agents are described above. In preferredembodiments, therapeutic agents for the treatment of Crohn's disease areadministered to the subject. Exemplary Crohn's disease therapeuticagents are anti-inflammatory agents such as agents comprisingmesalamine, immunosuppressive agents such as 6-mercaptopurine andazathioprine; biological agents such as infliximab (REMICADE®),antibiotics, and antidiarrheal agents such as diphenoxylate, loperamide,and codeine.

In embodiments wherein allogeneic stem cells are used, supportivetreatment may be required. For example, immunosuppressants may beadministered before, during and/or after treatment to prevent GVHD,according to methods known in the art. Prior to administration, thecells may also be modified to suppress an immune reaction from thesubject to the cells or vice-versa, according to methods known in theart.

The dosage of any therapeutic agent will vary depending on the symptoms,age and body weight of the patient, the nature and severity of thedisorder to be treated or prevented, the route of administration, andthe form of the agent. Any of the subject formulations may beadministered in a single dose or in divided doses. Dosages for thetherapeutic agents may be readily determined by techniques known tothose of skill in the art or as taught herein. Also, mixtures of morethan one therapeutic agent may be administered, or multiple therapeuticagents administered in separate compositions.

Therapeutic agents can be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally, orvia an implanted reservoir. The term parenteral as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, andintracranial injection or infusion techniques.

The precise time of administration and amount of any particular agentthat will yield the most effective treatment in a given patient willdepend upon the activity, pharmacokinetics, and bioavailability of aparticular compound, physiological condition of the patient (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage and type of medication), route ofadministration, and the like. The guidelines presented herein may beused to optimize the treatment, e.g., determining the optimum timeand/or amount of administration, which will require no more than routineexperimentation consisting of monitoring the subject and adjusting thedosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. Treatment, includingsupplement, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters, the first such reevaluation typicallyoccurring at the end of four weeks from the onset of therapy, andsubsequent reevaluations occurring every four to eight weeks duringtherapy and then every three months thereafter. Therapy may continue forseveral months or even years, with a minimum of one month being atypical length of therapy for humans. Adjustments to the amount(s) ofagent administered and possibly to the time of administration may bemade based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The combined use of several therapeutic agents may reduce the requireddosage for any individual component because the onset and duration ofeffect of the different components may be complimentary. In suchcombined therapy, the different active agents may be delivered togetheror separately, and simultaneously or at different times within the day.

Toxicity and therapeutic efficacy of subject compounds may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ and the ED₅₀. Compositions thatexhibit large therapeutic indices are preferred. Although compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets the agents to the desired site in order toreduce side effects.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany therapeutic agent or alternatively of any components therein, liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For agents of the present invention, the therapeuticallyeffective dose may be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information may be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5. Kits

In other embodiments, the invention contemplates kits including theadipose tissue-derived stromal stem cell-containing compositions andoptionally instructions for their use. Kits comprising thepharmaceutical compositions and biomaterials of the present inventionare also within the scope of the invention. Kit components may bepackaged for either manual or partially or wholly automated practice ofthe foregoing methods. Such kits may have a variety of uses, including,for example, therapy, repair, preparation of biomaterials and otherapplications.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Preparation of Stem Cells from Lipoaspirates with ImprovedHomogeneity

Adipose tissue was obtained by liposuction, under local anaesthesia andgeneral sedation. A hollow blunt-tipped cannula, was introduced into thesubcutaneous space through a small incision (less than 0.5 cm indiameter). With gentle suction, the cannula was moved through theadipose tissue abdominal-wall compartment for mechanical disruption ofthe fatty tissue. A saline solution and the vasoconstrictor epinephrinewere injected into the adipose tissue compartment to minimize bloodloss. In this way, 80 to 100 ml of raw of lipoaspirate were obtainedfrom each patient to be treated.

The raw lipoaspirate was washed extensively with sterilephosphate-buffered saline (PBS; Gibco BRL, Paisley, Scotland, UK) toremove blood cells, saline and local anaesthetic. The extracellularmatrix was digested with a solution of type II collagenase (0.075%;Gibco BRL) in balanced salt solution (5 mg/ml; Sigma, St. Louis, USA)for 30 min at 37° C. to release the cellular fraction. Then thecollagenase was inactivated by addition of an equal volume of Dulbecco'smodified Eagle's medium (DMEM; Gibco BRL) that contained 10% fetalbovine serum (FBS; Gibco BRL). The suspension of cells was centrifugedat 250×g for 10 min. Cells were resuspended in 0.16 M NH₄Cl and allowedto stand for 10 min at room temperature (RT) for lysis of erythrocytes.The mixture was centrifuged at 250×g, and cells were resuspended in DMEMplus 10% FBS and 1% ampicillin/streptomycin mixture (Gibco, BRL) andthen they were plated in 100-mm tissue culture dishes at a concentrationof 10-30×10³ cells/cm².

Cells were cultured for 24 h at 37° C. in an atmosphere of 5% CO2 inair. Then the dishes were washed with PBS to remove non-adhering cellsand cell fragments. The cells were maintained in culture in the samemedium and under the same conditions until they reached approximately80% confluence, with replacement of the culture medium every 3 to 4days. Cells were then passaged with trypsin-EDTA (Gibco BRL) at adilution of 1:3 which corresponds to a cell density of approximatelyabout 5-6×10³ cells/cm². For transplantation, we used cells betweenpassages 1 and 3, with cells having been passaged more than twice beingpreferable in order to isolate a cell population with high homogeneity.Cell characterization was performed using cells at passages 1 to 9.

Example 2 Characterization of Stem Cells from Lipoaspirates withImproved Homogeneity

To characterize the cells by immunofluorescence staining, cells wereplated at low density in DMEM plus 10% FBS on glass cover slips in24-well plates. For immunohistochemistry studies, cells were washed withPBS and fixed in acetone for 10 min at −20° C. For staining of a-actin,cells were fixed in 4% paraformaldehyde for 10 min at RT. After blockingwith a PBS that contained 4% goat serum and 0.1% Triton X-100, cellswere incubated at 4° C. overnight with primary antibodies against thefollowing cell markers at the indicated dilutions [(i) alpha-actin;Dako, Glostrup, Denmark; 1/50; (ii) vimentin; Sigma, St. Louis, USA;1/200; (iii) CD 90; CYMBUS, Biotechnology LTD, Chandlers Ford, Hants,UK; 1/50; (iv) Factor VIII; Dako; 1/100; (v) CD 34; Chemicon, Calif.,USA; 1/100; (vi) c-Kit; Chemicon; 1/100; (vii) desmin; Dako; 1/100;(viii) cytokeratin; Dako; 1/100 and (ix) S-100; Dako; 1/50]. Then cellswere incubated with the appropriate Fluorescein isothiocyanate(FITC)-conjugated or Tetramethylrhodamine isothiocyanate chloride(TRITC)-conjugated second antibodies (Sigma; 1/50) for 45 min at RT. Fornegative controls we omitted the primary antibodies. Nuclei werecounterstained with 4′,6-diamidino-2-phenylindole (DAPI). Cells werethen mounted in Mobiglow (MoBiTec, Gottingen, Germany) and observed withan epifluorescence microscope Eclipse TE300 (Nikon, Tokyo, Japan). Ineach case, we determined the numbers of immunopositive cells indifferent fields and compared them to the numbers of stained nuclei.Randomly selected fields were exported to a computer (MacIntosh G3;Apple Computer Ink., Cupertino, Calif., USA) through a Spotl camera(Diagnostic Instruments Inc., Tampa, Fla., USA). Human aortic smoothmuscle cells, human umbilical vein endothelial cells (HUVEC) and humansynovial fibroblasts were used as positive controls for immunostainingwith the various antibodies.

At passage 1, a high percentage (90-95%) of adipose-derived stromal stemcells expressed vimentin, a marker of mesenchymal cytoskeletal cells(FIG. 1). Expression of vimentin was maintained at the same level up toand including passage 9. Levels of other markers fell, however, withtime. For example, a-actin, which was found in 17% of LPA-derived cellsat passage I was no longer detectable at passage 7. The marker ofendothelial cells, von Willebrandt factor (Factor VIII), and CD34, whichis also found on the surface of endothelial cells, were only detected atpassages 1 through 3 (7% and 12% immunopositive cells, respectively). Bycontrast, the expression of c-Kit (CD 117), a marker of cellproliferation, increased with time, with 99% immunopositive cells frompassage 4 onwards (FIG. 2). The fibroblast marker CD90, initiallyexpressed in approximately 80% of LPA-derived cells, was found in 99% ofcells from passage 6 (FIG. 3). No expression of the neuroectodermalmarker S100 or the ectodermal marker keratin was observed in any of theLPA-derived cells at any time. The change in observed markers as thenumber of passages increases indicates an increase in the homogeneity ofthe cell preparation obtained.

To quantitate cell growth, cells were plated in 24-well plates at aconcentration of 5×10³ cells/cm². After cells had attached to thesubstratum (3 h), the culture medium was replaced by DMEM supplementedwith 1% antibiotics plus 0.5%, 2%, 5% or 10% FBS. As positive controlsfor testing of each batch of serum, human synovial fibroblasts were alsocultured and their growth rates determined. Medium was replaced everytwo days. At 24-h intervals, cells were fixed with 1% glutaraldehyde andthe number of cells per well was determined, after nuclear staining withcrystal violet, by monitoring absorbance at 595 nm. A standard curve wasconstructed to establish the relationship between cell number per welland absorbance at 595 nm (r²=0.99).

Viable adipose tissue-derived stromal cells were successfully isolatedand cultured from all seven lipoaspirates (LPAs). These cells were grownin culture and passaged at 7 to 10 day intervals. In some cases, cellswere cryopreserved and thawed prior to implantation. The growth rate ofadipose tissue-derived stromal stem cells (ADSC) depended on the serumconcentration, with maximal proliferation between 5% and 10% FBS (FIG.4). The mean population-doubling time at these concentrations of serumwas 37.6±0.6 h, which did not differ significantly from thepopulation-doubling time of human synovial fibroblasts cultured underthe same conditions (35.6±1.4 h; p>0.05; t-test; results from threeindependent experiments).

In order to analyze the cells in a more standardized and less subjectivemanner, the cells were also subjected to Fluorescence Activated CellSorter (FACS) analysis. In general, the flow cytometry analysis permitsthe detection of surface antigens by antibodies, which are directly(covalently) or indirectly (secondary fluorescent-labeled antibody)linked to a fluorescent marker. On the other hand, the above describedimmunohistochemical analysis demanded permeabilization of the cells andthe subsequent staining with antibodies. Thus, the latter requires anindividually optimized protocol depending on target protein andantibody. Moreover, due to the permeabilization of the cell membrane, itis not possible to distinguish between internal (non-membrane bound) andextracellular marker proteins. That is, with an immunohistochemicalanalysis it is possible to know if a protein marker is being expressedbut it is not possible to distinguish if it is being expressed at thecell surface or intracellularly.

The protocol used in the immunocytometry for the detection of surfaceantigens is standardized, and only requires appropriate negativecontrols. Further, the FACS analysis allows an evaluation of thepercentage of positive cells (cells expressing the surface antigen), andthe level of expression (few or many surface antigens on one cell).These evaluations are only of subjective nature usingimmunohistochemistry, and may vary from experiment to experiment, whichdoes not occur with the FACS analysis.

Such immunophenotypic characterization of the cells may be performed onfreshly isolated cells and after periods of cultures, for example, atday 7, after 4 weeks and after 3 months of culture. The analysis ofsurface markers at different times allows the assessment of thehomogeneity of the phenotype during culturing. Examples of this analysisand data demonstrating the phenotype obtained from samples obtained from3 healthy donors from zero to three months of culturing are described atlength in U.S. patent application Ser. No. 11/065,461, filed on Feb. 25,2005, which is incorporated herein by reference.

After isolation by the above described method, the adipose-derivedstromal stem cells from one of the patients were characterized infunction of the presence/absence of a series of surface markers. To dothis, the expression of the following surface markers was monitored byflow cytometry:

Integrin: CD11b, CD18, CD29, CD49a, CD49b, CD49d, CD49e, CD49f, CD51,CD61, CD104.

Hematopoietic markers: CD3, CD9, CD10, CD13, CD16, CD14, CD19, CD28,CD34, CD38, CD45, CD90, CD133, glicoforine.

Growth factor receptors: CD105, NGFR.

Extracellular matrix receptors: CD15, CD31, CD44, CD50, CD54, CD62E,CD62L, CD62P, CD102, CD106, CD146, CD166.

Others: CD36, CD55, CD56, CD58, CD59, CD95, HLA-I, HLA-II,β2-microglobuline.

The cells to be characterized were collected by means of gentledigestion with trypsin, washed with PBS and incubated for 30 minutes at4° C. with fluorescein (FITC) or phycoerythrin (PE) labeled antibodymarkers against each one of the surface markers to be analyzed. The cellmarkers were washed and immediately analyzed using the Epics-XLcytometer (Coulter). As controls, cells stained with unspecificantibodies of the corresponding isotopes labeled with FITC or PE wereused.

From the analysis of the profile of expression of surface markers (FIG.7A/7B), the criteria used to determine which markers define the cellpopulation and allow it to be identified and differentiated with respectto other types of cell were the following:

-   -   1. Discard those markers that vary from one sample to the other        or over time during culturing in the experimentation done with        healthy donors' adipose-derived stromal stem cells in the U.S.        patent application Ser. No. 11/065,461, filed on Feb. 25, 2005,        which is incorporated herein by reference.    -   2. Select the markers as a function of their biological        relevance, discarding markers characteristic of specific cell        types (for example, CD3 is a marker exclusive to lymphocytes).

Applying these criteria, the multipotent stem cell population ischaracterized as being positive for CD9+, CD10+, CD13+, CD29+, CD44+,CD49A+, CD51+, CD54+, CD55+, CD58+, CD59+, CD90+ and CD105+; and lackingexpression of CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102,CD104, CD106 and CD133.

Example 3 Preparations of Stem Cells Comprising Fibrin Glue for Use inTreating Fistula

For clinical use, the cells as prepared above may be used after three orfewer passages (FIG. 3), but are preferably used after two or morepassages as described above to afford a cell preparation with higherhomogeneity. Cell cultures for clinical use were trypsinized for 3 minat 37° C. Trypsinization was stopped by addition of DMEM plus FBS, andthe suspension was centrifuged at 110×g for 5 min. Cells were washed inPBS and the suspension was centrifuged again at 150×g for 5 min. Cellwere resuspended at between 3 and 30×10⁶ cells/ml in 1 to 2 ml ofRinger's lactate solution and put in a suitable syringe. Human serumalbumin (HSA) may optionally be added to the Ringer's lactate solution.

In certain cases, half of the cells were resuspended in the thrombincomponent of a fibrin glue kit (TISSUCOL® Duo 2.0; Baxter, Madrid,Spain) prior to combination of the kit's two components, in an attemptto improve the obturation of the fistulae' tracts. The use of fibringlue to fill a fistula opening is known in the art; however, it is notefficient as a standalone treatment for fistula. The addition of fibringlue to the adipose tissue-derived stromal stem cell-containingcompositions described herein serves to retain cells locally, and wehave observed that the cells grow well inside fibrin glues and gels.

Example 4 Improved Surgical Procedure to Repair Fistula UsingPreparations of Stem Cells from Lipoaspirates

We conducted a phase I clinical trial designed to test the feasibilityand safety of autologous stem cell transplantation using theabove-described adipose tissue stromal stem-cell compositions fortreatment of Crohn fistulae. The protocol was approved by the ClinicalTrial and Ethics Committee of La Paz Hospital on Apr. 12, 2002, and adetailed informed-consent form was generated to be signed by patients.The Ethics Committee was kept informed about the progress of the studythroughout the clinical trial.

Methods

The patients were selected according to the following inclusioncriteria: more than 18 years of age; diagnosis of Crohn's disease atleast five years prior to the trial; presence of one or more complexCrohn fistulae (enterocutaneous fistula, suprasphincteric fistula and/orrecto-vaginal fistula) that had been unresponsive to medical treatmentand unsuccessfully treated by classical surgery at least twice; andagreement to participate, with signature of the informed-consent form.The exclusion criteria were as follows: Failure to meet inclusioncriteria; mental handicap; extreme thinness; allergy to localanaesthetics; prior diagnosis of cancer; and AIDS.

Five patients (nos. 001-005) were enrolled in the study. There werethree men and two women, and the average age was 35.1±2.4 years (range:31.2 to 37.5 years). Nine cell implants were performed: three inrecto-vaginal fistulae; five in enterocutaneous fistulae (four differentfistulae in one patient; and one in a suprasphincteric perianal fistula.All enterocutaneous fistulae had low flow—less than 50 cc per day—andwere located in the abdominal wall (Table 1). No patient was treatedwith Total Parenteral Nutrition, Remicaid or Octreotride concurrentlywith this procedure. Patients 001 and 002 required two liposuctionprocedures because, after the first liposuction, no stem cells survivedcryopreservation.

One patient was excluded due to bacterial contamination of culturedcells. We inoculated nine fistulae in four patients with autologousadipose tissue-derived stromal stem cells (ADSC) at passage three orearlier. Eight inoculated fistulae were followed weekly for at leasteight weeks. In six fistulae, the external opening was covered withepithelium at the end of week 8 and, thus, these fistulae wereconsidered healed (75%). In the other two fistulae, there was onlyincomplete closure of the external opening, with a decrease in outputflow (not healed; 25%). No adverse effects were observed in any patientat the end of the follow-up period (at least six months and no more thantwo years).

In the case of enterocutaneous fistulae, all tracks were deep scraped.In the case of recto-vaginal fistulae, we used a vaginal approach, withdetachment of the posterior vaginal wall. The gap was completelyseparated and the rectal opening was closed with 3/0 absorbablestitches. The rectal mucosa had been damaged by Crohn's disease and wasextremely fragile. In the case of perianal fistulae, the main track wascored out and the rectal hole was closed with 3/0 absorbable stitchesthrough the sclerotic mucosa.

Using a needle, in cases of enterocutaneous fistula, we injected cellsinto the wall of the track. In the cases of recto-vaginal and perianalfistulae, we injected cells into the rectal mucosa, close to the suturedinternal opening. In all cases, we observed a fluid-filled blister onthe area of the injection after the injection (FIG. 5). The number ofinjected cells ranged from 3 to 30×10⁶, depending on the growth of thecultured cells (FIG. 3).

The time from the beginning of preparation of the inoculum to the end ofthe injection was less than 90 minutes in all cases. In the case ofenterocutaneous fistulae, tracks were filled with fibrin glue and thenthe skin was sutured. In case of recto-vaginal fistulae, an advancementvaginal flap was constructed. When accessory tracks were detected, theywere also filled with fibrin glue.

No bandages were applied post-operatively. Liquid intake was initiatedtwelve hours after the procedure and solid food six hours afterwards.One to three days after surgery, the patient was dismissed and follow-upvisits were scheduled at the out-patient clinic.

Two histopathological samples were obtained. One specimen (patientnumber 002) was obtained from the area of an enterocutaneous fistula (7months after implant #2 and 10 days after implant #3). The otherspecimen (patient number 001) was obtained from the rectovaginal wall,one year after the first implant, (implant #1), during the surgicalprocedure associated with implant #6. The specimens were embedded inparaffin, sectioned, stained with haematoxylin and eosin, and evaluated.

Weekly follow-up was scheduled for eight weeks after surgery. Patientswere considered healed when a total epithelialization of the externalopening was evident after eight weeks, independently of priorobservations. After eight weeks, there was a monthly follow-up for atleast six months and not more than two years.

Results

Five patients were included in the study and seven liposuctions wereperformed (FIG. 3). Patient number 003 was eliminated from the trialduring the implant procedure as a result of the discovery ofcontamination by Gram-positive bacteria of the cultured lipoaspiratedcells. The bacteria were identified as Oerkoviu xanthineolytica. Anenterocutaneous fistula in patient 002 was eliminated from the studybecause of emergency abdominal surgery for a new enterovesicular fistulathat had resulted in acute sepsis. The laparotomy required the resectionof the implant area. Therefore, we could not adhere to the minimumeight-week follow-up schedule in this case.

Nine fistulae from four patients were inoculated with ADSC after threeor fewer passages (FIG. 3). Eight fistulae were considered suitable forretention in the study and followed for at least eight weeks (FIG. 3).In six fistulae, the external opening had epithelialized by week 8 andthese fistulae were considered healed (75%) (FIG. 6). The other two hadonly incomplete closure of the external opening, with a decrease inoutput flow, as reported by the patients (25%; not healed; FIG. 3).There was no direct relationship between the number of cells injected orculture time and success of the procedure. There was also no directrelationship between the patient's gender or age and healing.Subsequence studies we have done indicate that an initial dose of 20×10⁶cells is suitable. We have determined that a second dose of 40×10⁶ cellsmay be used in the event that the first dose fails. Higher cell dosesare preferred because we have observed that higher cell numbers have abetter therapeutic effect in tissue repair.

Surgical and implantation procedures were performed without additionaltechnical difficulty in all nine treated fistulae. No immediate adversereactions (e.g., anaphylaxis, allergic reactions) were observed in anyof the cases studied.

Two histopathological samples were obtained seven months(enterocutaneous fistula) and one year (recto-vaginal fistula) aftersurgery. No cytological transformation was detected in a complete seriesof histopathological sections.

Discussion

In a previous report, we described the successful cell-based treatmentof a young woman with a recurrent recto-vaginal fistula that had beenunresponsive to medical treatment. Thus, we designed the present phase Iclinical trial to evaluate the feasibility and safety of such autologousadipose tissue stromal stem-cell transplantation (with improvements inthe original protocol) for the treatment of unresponsive Crohn fistulae,as well as to test the use of adipose tissue stromal stem-cells inconjunction with a fibrin glue.

We chose adipose tissue as the source of stem cells because of theircapacity for myogenic differentiation and the fact that fistulae respondwell to muscle transplants. Moreover, liposuction fat is available inlarge quantities and can be harvested with minimal adverse effects onthe patient. Other groups have used bone marrow-derived stem cells but,in such cases, a cell-mobilization procedure is required that can bedangerous to some patients, such as those with a myocardial infarction.In our study, all liposuction procedures yielded a clinically usefulnumber of cells with characteristics of stem cells.

We followed our patients according to the program scheduled, and weobserved a complete healing in 6 of 8 procedures. It is important tonote that Crohn's disease provides the worst conditions for a surgicalapproach to fistulae because of the fragility of the tissue and theenormous problems associated with healing in these patients. Ourpatients were chosen because they had been unresponsive to medicaltreatment and at least two previous surgical procedures but ourtreatment seemed to be quite effective. Nevertheless, new outbreaks ofCrohn's disease may still produce new fistulae in any given patient thatwill need to be treated again using the cryopreserved autologous cellsfrom that patient.

The biological mechanism that underlies the therapeutic success of ADSCtransplantation is unknown. Stem cells might differentiate intoconnective, muscle or scar tissue. Alternatively, secretion of growthfactors by the stem cells might facilitate wound healing. We saw typicalscar tissue in the histopathologically examined fistulae, but we have noway of distinguishing transplanted from local connective-tissue cells.We observed a complete healing in 75% of cases using our treatment.

REFERENCES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

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EQUIVALENTS

The present invention provides, among other things, methods andcompositions for treating and preventing fistula. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The appended claims are not intended toclaim all such embodiments and variations, and the full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. (canceled)
 2. A method of treating a fistula in a subject, comprisingdelivering a population of cells comprising adipose tissue-derivedstromal cells to the subject, wherein the adipose tissue-derived stromalcells are allogeneic to the subject.
 3. The method of claim 2, whereinthe population comprises at least 10×10⁶ adipose tissue-derived stromalcells.
 4. The method of claim 2, wherein the adipose tissue-derivedstromal cells have been passaged at least six times.
 5. The method ofclaim 2, wherein less than 5% of the cells in the population express theCD34 marker.
 6. The method of claim 5, wherein at least 50% of the cellsin the population express the CD44 marker.
 7. The method of claim 2further comprising deep scraping of the fistula prior to delivering theadipose derived tissue-derived stromal cells and filling said fistulawith an adhesive before, during or after delivering the population ofcells.
 8. The method of claim 7, wherein the population comprises atleast 10×10⁶ adipose tissue-derived stromal cells that are mixed withthe adhesive before delivering the population of cells.
 9. The method ofclaim 7, wherein said adhesive is a fibrin glue or gel.
 10. The methodof claim 7, wherein said adhesive further comprises a compositioncomprising adipose tissue-derived stromal cells, wherein less than 5% ofthe cells in the composition express the CD34 marker.
 11. The method ofclaim 10, wherein at least 50% of the cells in the composition expressthe CD44 marker.
 12. The method of claim 2, wherein said method furthercomprises: delivering a second dose of at least 20×10⁶ adiposetissue-derived stromal cells.
 13. The method of claim 2, wherein thepopulation comprises at least 20×10⁶ adipose tissue-derived stromalcells.
 14. The method of claim 13, wherein said method furthercomprises: delivering a second dose of at least 40×10⁶ adiposetissue-derived stromal cells.
 15. The method of claim 2, wherein thefistula is selected from the group consisting of an anorectal fistula,enteroenteral fistula, enterocutaneous fistula, rectovaginal fistula,vesicovaginal fistula, fistula-in-ano, fecal fistula, arteriovenousfistula, biliary fistula, cervical fistula, craniosinus fistula,enterovaginal, gastric fistula, metroperitoneal fistula perilymph,pulmonary arteriovenous fistula, umbilical fistula, andtracheoesophageal fistula.
 16. The method of claim 2, further comprisingadministering a therapeutic agent to said subject.
 17. The method ofclaim 16, wherein said therapeutic agent is administered locally to thefistula.
 18. The method of claim 16, wherein said therapeutic agent isadministered systemically to said subject.
 19. The method of claim 16,wherein said therapeutic agent is selected from the group consisting ofan anti-inflammatory agent, an immunosuppressive agent, a biologicalagent, an antibiotic and an antidiarrheal agent.
 20. A method oftreating a wound in a subject, comprising: (a) closing the wound with asuture and (b) delivering a composition comprising at least 10×10⁶adipose tissue-derived stromal cells to the closed sutured wound,wherein the adipose tissue-derived stromal cells are allogeneic to thesubject.
 21. The method of claim 20, wherein less than 5% of the cellsin the composition express the CD34 marker.
 22. The method of claim 21,wherein at least 50% of the cells in the composition express the CD44marker.
 23. The method according to claim 20, wherein the wound is afistula selected from the group consisting of anorectal fistula,enterorectal fistula, enterocutaneous fistula, rectovaginal fistula andvesicovaginal fistula.
 24. A method of treating a fistula in a subjectcomprising the steps of: (i) deep scraping of at least one fistula track(ii) closing the internal hole of the scraped track with a suture (iii)delivering composition comprising at least 10×10⁶ adipose tissue-derivedstromal cells to the closed sutured internal hole, wherein the adiposetissue-derived stromal cells are allogeneic to the subject.